Non-Diabetic Kidney Disease in Type 2 Diabetes Mellitus: A Changing Spectrum with Therapeutic Ascendancy

Background and objectives: Owing to changing epidemiology and therapeutic practices, a change in the spectrum of renal involvement in Type-2 diabetes mellitus (T2DM) has also been noted. The treatment of non-diabetic kidney disease (NDKD) differs from diabetic kidney disease (DKD) and the reversibility of NDKD in many cases to normal, prompts biopsy for rapid and accurate diagnosis. Data are scarce on kidney biopsy findings in T2DM. Study design & setting: In this observational study, we prospectively collected the data of kidney biopsies of patients aged ≥ 18 years with T2DM admitted between 1 August 2005 and 31 July 2022. The clinical, demographic and histopathological data were evaluated. The spectrum of kidney involvement in the form of DKD and/or NDKD was studied. The impact of these findings with the use of drugs retarding disease progression was also analyzed. Results: A total of 5485 biopsies were performed during the study period and of these 538 patients had T2DM. The mean age of the study population was 56.9 ± 11.5 years and 81% were males. The mean duration of DM was 6.4 ± 6.1 years. Diabetic retinopathy (DR) was noted in 29.7%. The most common indication for biopsy was an acute rise in creatinine (147, 27.3%). Amongst the 538 diabetic patients who underwent biopsy, histological features only of DKD were noted in 166 patients (33%), NDKD alone in 262 (49%) and NDKD with DKD lesions in 110 (20%). On multivariate analysis, duration of DM less than 5 years, absence of CAD, absence of DR, oliguria at presentation, an acute rise in creatinine and low C3 were associated with NDKD. Conclusions: The prevalence of NDKD among diabetics and ATIN in particular might be on an increasing trend in the current era of changing T2DM epidemiological patterns. The use of anti-pro-teinuric agents was associated with lesser degrees of histopathological chronicity in T2DM.


Introduction
Diabetes Mellitus (DM) affects the kidney either in the form of diabetic kidney disease (DKD), non-diabetic kidney disease (NDKD), or both overlapping [1]. Despite an increasing prevalence of type-2 diabetes mellitus (T2DM), the overall prevalence of DKD remained stable [2]. Apart from better glycemic control by early diagnosis, this could be due to the improved understanding of the pathogenesis, at least of the hemodynamic mechanisms, which led to the discovery and use of newer therapeutic agents, including renin-angiotensin-aldosterone system blockers (RAASB) and sodium-glucose transporter-2 inhibitors (SGLT2I). Recently, an increasing prevalence of NDKD in diabetics has been reported [3], which may be due to the aging population, higher exposure to infections, increasing incidence of monoclonal gammopathies and other malignancies [3,4]. Consequently, an increased prevalence of NDKD and non-proteinuric DKD is expected to rise in T2DM patients. More importantly, many NDKD conditions are treatable and complete reversibility is possible, which warrants a timely biopsy, accurate diagnosis and prompt treatment.
In the face of these epidemiological changes, histological and clinical characteristics are also likely to change; hence a re-evaluation of kidney disease in T2DM is implicated. Sharma SG et al. found an increasing prevalence of NDKD and a changing spectrum of NDKD [4]. For instance, acute tubular necrosis (ATN) was the most common type of NDKD found in contrast to IgA Nephropathy, as noted in an earlier meta-analysis [5]. Moreover, due to relatively poor glycemic and blood pressure control, delayed diagnosis and higher infection rates in the developing world, the NDKD spectrum differs from western studies and cannot be extrapolated to the Indian population [6]. Given the huge burden of diabetes in India, which accounts for nearly one-fifth of the diabetic population in the world, the burden of diabetes-related kidney diseases is also expected to be high [7]. Hence, knowing the spectrum of kidney diseases in T2DM is essential for optimal management. The current study attempts to analyze clinicopathological characteristics of various kidney diseases along with differences in findings with RAASB and SGLT2I treatment in patients with T2DM admitted for a kidney biopsy.

Materials and Methods
In this study, we prospectively collected data of all kidney biopsies performed in the department between the study period from 1 August 2005, till 31 July 2022. Written and informed consent was obtained from each patient for the kidney biopsy and inclusion into the study at time of the kidney biopsy. The study was conducted as per the declaration of Helsinki. The institutional ethics committee has approved the study.
We separated the biopsy details and clinical features at the presentation of the patients on the electronic records of the hospital information system of this public sector tertiary care institute. All patients had standard indications for biopsies, i.e., absence of retinopathy, presence of active sediments, red blood cells/cast in urine, heavy proteinuria on first presentation and sudden deterioration in degree of proteinuria, rapid decline in GFR as per standard guidelines [8,9]. Patients with Type-1 DM and pregnant and lactating females were excluded.
The indications of biopsies were prospectively noted. The data included demographic details (age, gender, duration of diabetes), associated comorbidities, diabetes-related microvascular and macrovascular complications, type of renal presentation (Acute kidney injury/Acute on CKD/Rapidly progressive renal failure (RPRF)), eGFR at presentation, dialysis dependency, proteinuria, presence of microscopic hematuria, hypoalbuminemia, hypercholesterolemia, hypocomplementemia and the physician's indication of biopsy. Therapeutic parameters were collected, including therapy with RAASB, SGLT2I, insulin, other anti-hypertensives and glycemic control before the biopsy. The details of standard definitions, disease classification and the disease stages are described in the supplementary file (SI). The definitions of DM, acute kidney injury (AKI), acute on chronic kidney disease (AKI on CKD), rapidly progressive renal failure (RPRF) and rapid decline in GFR were considered as per the standard diagnostic criteria shown in previous studies and briefed in the supplementary file (SI) [10][11][12][13][14][15][16]. Similarly, comorbidities such as hypertension, coronary artery disease, cerebrovascular and peripheral vascular disease, diabetic retinopathy and diabetic neuropathy were defined as per the standard disease definitions [17][18][19][20][21][22].
All biopsy samples were processed for light microscopy, immunofluorescence and electron microscopy if indicated [19]. Two pathologists, VA and MJ, reviewed histological changes in DKD, NDKD and DKD plus NDKD, as described in the supplementary file (SI L, I M). Disputed findings were resolved by mutual agreement.
The DKD and various NDKD were classified as per the standard diagnostic criteria shown in the supplementary file (SI) [23][24][25][26][27][28][29][30]. Patients were categorized into three groups-DKD, NDKD and NDKD plus DKD groups. Briefly, the presence of features such as Kimmelstiel-Wilson (KW) lesions, mesangial expansion, capsular drops, fibrin caps and the absence of any other features of NDKD was defined as DKD. Pure NDKD was defined by the presence of predominantly vasculopathy, interstitial fibrosis, tubular atrophy and/or specific glomerular changes in the absence of classical changes of DKD. The histological pictures of the presence of both were defined as DKD plus NDKD. The clinical predictors of NDKD were determined. The clinical and histological findings were compared between DKD, NDKD and DKD plus NDKD. We also compared the histopathological features of patients on RAASB and/or SGLT2I versus those not on the same drugs.

Statistical Analysis
Statistical analysis was performed by IBM, SPSS software, version 25. The Shapiro-Wilk test was used to determine the normality of the distribution of data values. All the continuous data were expressed in the form of mean and standard deviation if data were normally distributed, and the non-normally distributed data were expressed in the median and interquartile range. Categorical data were expressed in percentages. The Chi-square test or Fischer's exact test was used to compare the categorical values between the groups, as per the application required. Student's t-test was used to compare the mean values and continuous variables if it was normally distributed; otherwise, Mann-Whitney's U-test was used. A stepwise multivariate logistic regression analysis was used to analyze the clinicopathological factors predicting NDKD. A p-value of <0.05 was considered significant.

Results
A total of 5485 biopsies were performed during the study period; of these, 538 (mean age 56.9 ± 11.5 years and 436 (81%), males) patients had T2DM (Figure 1). The baseline characteristics of the study population are as shown in Table 1. The mean duration of T2DM was 6.4 ± 6.1 years. Coexisting hypertension (HTN) was seen in 419 (78%) patients and of these only 244 (45.4%) patients had HTN onset after the onset of T2DM. Among the diabetes-related other end-organ changes, DR was most common, seen in 160 patients (29.7%); of these, most (122 patients, 76.2%) had non-proliferative DR. A total of 39.8% had nephrotic syndrome at presentation, 34% patients had AKI (including acute on CKD) and 7.8% had RPRF at presentation. The median eGFR at presentation was 17.2 (IQR: 9.5-41.7) mL/min/1.73 m 2 and 29.2% had dialysis requiring renal failure. The mean proteinuria was 4.7 ± 3.9 g/day, with microscopic hematuria in 46.3% of patients.

Histological Comparison between DKD and NDKD
The histopathological features are shown in Supplementary Tables S1 and S2. Comparing the vascular and tubulointerstitial changes on renal biopsy between the two groups (Supplementary Table S1), a significantly higher proportion of NDKD patients had no IFTA (15.8% vs. 1.8%, p = 0.001) than DKD patients. A higher proportion of DKD patients had a moderate degree of IFTA (47% vs. 29.8%, p = 0.001), suggesting a higher degree of chronicity among the patients with DKD ( Figure 4C). Among the vascular changes, both afferent and efferent arteriolar hyalinosis was noted in 84.3% of DKD patients compared to 23.9% of NDKD patients (p = 0.001). The majority of patients in the NDKD group had either no hyalinosis (38.9% vs. 7.2%, p = 0.001) or only afferent arteriolar hyalinosis (37% vs. 8.4%, p = 0.001) as compared to DKD patients. On the evaluation of the immunofluorescence examination findings, 46.4% of patients with DKD had no immune deposits compared to 11.5% among the NDKD group (p = 0.001). The majority of NDKD patients had C3 deposits (31.4% vs. 15%, p = 0.001).

Histological Comparison between DKD and NDKD plus DKD
The comparison of histological classes of DKD and the diabetes-related glomerular changes in the DKD-only group and those with NDKD + DKD are shown in Supplementary  Table S2. There were no differences in the distribution of various classes of DKD. Eight patients (4.8%) with DKD and two patients (1.8%) with NDKD plus DKD had no diabetesrelated glomerular changes on light microscopy (p = 0.19). They had diabetes-related GBM thickening on EM only ( Figure 4D). Nearly half of the patients (49%) among the NDKD plus DKD group had GBM thickening compared to 29.5% among the DKD patients (p = 0.003). Likewise, a higher percentage of patients with NDKD plus DKD had severe mesangial expansion (22.7% vs. 9.3%, p = 0.001) and K-W lesions (47.2% vs. 33.1, p = 0.03), compared to DKD only group. Most of the patients with only DKD had mild mesangial expansion (70.4% vs. 40.9%, p = 0.001).

Discussion
In this study, we analyzed clinical, laboratory and pathological features in 538 T2DM patients who underwent kidney biopsies for various indications. This accounted for onetenth of all the biopsies performed at our center. While nearly one-third had only DKD, 70% had NDKD, of which 50% had pure NDKD and 20% had NDKD with DKD. Given the high pre-test probability of NDKD, a higher percentage of NDKD was expected, reiterating the findings of Sharma et al. [4]. On the contrary, nearly a third of diabetic patients with atypical clinical features eventually had DKD on biopsy.
We found DR only in 45% of patients with DKD compared to more than 60% in previous studies [31,32], which suggests the declining correlation between the two microvasculopathies, similarly evidenced by Pedro et al., who found DR in <30% of T2DM with DKD [33]. More importantly, up to 80% of the patients without DR eventually had NDKD. Similarly, many recent studies also showed that 70-80% of diabetic patients with NDKD had no DR [34,35]. Accordingly, "the renal-retinal relation" in T2DM may be better asserted, as the absence of DR may reasonably point towards NDKD. However, the presence of DR does not predict DKD, as also evidenced in other previous studies [36,37]. Similarly to this one no correlation between diabetic neuropathy and DKD was found in a previous study [38].
Coexisting macrovascular complications were noted in more than 10% of DKD patients. We observed lesser numbers of CAD in our study compared to prior studies, where about 60% of diabetic patients with CKD had CAD [39,40]. This could be due to the cross-sectional nature of the data retrieved at the time of the kidney biopsy and the lack of follow-up. Despite the good evidence that CKD in diabetes was associated with an increased CAD risk, whether DKD is associated with higher CAD risk compared to NDKD remains debatable. We found that, among diabetics with kidney failure, the presence of DKD, in particular, doubles the risk of CAD compared to NDKD (12% vs. 6%). Microscopic hematuria was observed in 40% of DKD patients, similar to prior studies in 20-40% of patients [41]. Nonproteinuric kidney disease was noted more in the NDKD group; only about 5% of DKD patients had a non-proteinuric phenotype in our study. Despite the increasing prevalence of non-proteinuric DKD phenotype currently, [42,43], less prevalence in our study could probably be because this phenotype might not have been considered for kidney biopsy. We also observed that two-thirds of patients with NDKD had diabetes duration of <5 years and up to 40% with T2DM had a pre-existent HTN, as observed in other studies [44,45].
Moreover, a significantly higher proportion of DKD patients had an onset of HTN after at least two years of diagnosis of T2DM. Our study emerged with strong clinical evidence that diabetes duration <5 years, absence of CAD, absence of DR, oliguria at presentation, an acute rise in creatinine and low C3 were predictors of NDKD on multivariate analysis. Microscopic hematuria, HbA1C level, age, gender, etc., were the other factors noted to be higher in NDKD patients, although non-significant on multivariate analysis.
Amongst NDKD, ATIN was the most common pathology in our study, in concordance with evidence in the past decade [4,46,47], unlike in prior studies, where glomerular lesions, such as IgAN and MGN, were more common NDKD pathologies [48,49]. A meta-analysis by Fiorentino et al. further supported our findings of the increasing prevalence of ATIN in recent studies [5]. The changing pathology of NDKD could be partially attributed to epidemiological changes, the aging population and increased infections and malignancy risk. Besides, nephrotoxic medications and herbal remedies exposures may also contribute to ATIN in a pre-existent state of diabetes-related vascular ischemia. The types of NDKD pathology may also depend on study inclusion criteria and indications for biopsy, besides demographic and genetic variations in the study population. We performed only clinically indicated biopsies on patients with a high suspicion of NDKD; therefore, outcomes may differ from research-indicated biopsies. Hence, as the most common indication for biopsy in our study, AKI, either with (23%) or without CKD (7%), ATIN and proliferative GN contributed to more than 50% of the NDKD pathologies. However, membranous nephropathy and IgA nephropathy were the common pathologies amongst non-AKI indications groups, in concurrence with previous studies. Our finding for NDKD pathologies emphasized the need for a high index of clinical suspicion and for kidney biopsies in the current period when evaluating renal dysfunction in diabetics.
The study's strength lies in the large sample size and prospective data collection during biopsy. Our study is plausibly the first to evaluate the clinicopathological findings predicting NDKD in the best possible way in a single study and conceivably the first to analyze the association between RAASB therapy and/or SGLT2I therapy with histopathological findings in both DKD and NDKD patients. Data on the impact of SGLT2I on histological findings are currently scarce. Major limitations are the lack of long-term follow-up data post-biopsy, and selection bias for indication biopsy remains unavoidable.

Conclusions
To conclude, the prevalence of NDKD among diabetics and ATIN, in particular, might be on an increasing trend in the current era of changing epidemiology and therapies for T2DM. Because of the treatable nature of the condition and its potential to accelerate progression with delayed diagnosis of renal dysfunction, a greater emphasis on timely diagnosis is required for this particularly vulnerable cohort. The spectrum of NDKD is vast and may be missed without a kidney biopsy.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jcm12041705/s1, Section S1. Standard working definitions and classifications, Table S1. Comparison of histopathological features, Table S2. Histopathological findings of DKD on renal biopsy-DKD versus NDKD plus DKD. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee of SGPGIMS (IEC code2012-120-IP-63) for studies involving humans.
Informed Consent Statement: Written informed consent was obtained from all study subjects involved in the study.

Data Availability Statement:
The data can not be public due to institution policy. However, it is available with the corresponding author and can be made available upon reasonable request.